The proposed work will further define the mechanisms controlling the proliferation of Leydig cell progenitors and their differentiation into mature Leydig cells. Previous morphological studies carried out by the Principal Investigator and others provide strong support for the hypothesis that the Leydig cell differentiates from a mesenchymal-like progenitor. In parallel with these studies, fractions of interstitial cells from testes of immature rats were isolated and cultured in vitro to further define the hormonal requirements for differentiation of Leydig cells from progenitor cells. This laboratory has routinely isolated a fraction of interstitial cells from the testes of 21-day-old rats. Those cells showed increased production of testosterone on day 3 in vitro only when cultured in the combined presence of luteinizing hormone (LH) and androgen. Taken together, the results indicate that the fraction of interstitial cells was enriched for Leydig cell progenitors, and that LH and androgen have key roles in stimulating the conversion of the progenitor cell into the testosterone-secreting Leydig cell. Studies delineated in this proposal will achieve the following Specific Aims. In Aim A we will evaluate the respective roles of LH, androgen, and two growth factors (insulin-like growth factor-I and interleukin-1) in proliferation of progenitor cells, immature, and adult Leydig cells. In Aim B we will study the mechanisms by which these factors stimulate differentiation of progenitor cells into immature Leydig cells, and immature into adult Leydig cells. In Aim C we will study the mechanisms by which these factors regulate the activities of testosterone biosynthetic and metabolizing enzymes during differentiation of progenitor cells into immature Leydig cells and immature into adult Leydig cells.